Central to the form and function of muscles are the myofibrils that provide the cytoskeletal structure and the contractile force that characterize skeletal muscles. The long-term goal of the proposed experiments is to understand the steps and proteins governing the assembly of myofibrils in skeletal muscle cells. The first strategy of our experimental approaches is to use microscopic techniques to analyze myofibril formation inside single living skeletal muscle cells transfected with probes expressing sarcomeric proteins linked to Green Fluorescent Proteins. Our second approach is focused on a molecular analysis of proteins interacting with the Z-bodies and Z-bands of the assembling myofibrils. The first specific aim will test the hypothesis that the formation of myofibrils in myotubes proceeds from premyofibrils to mature myofibrils, and requires nonmuscle myosin II and alpha-actinin molecules. We will use time-lapse fluorescence microscopy to analyze the assembly of sarcomeric subunits into myofibrils in cultures of living skeletal muscle cells. Three independent avenues will be used to block the activity or presence of the non-muscle myosin II in an effort to test the its role in the pathway from premyofibrils to mature myofibrils. Proteolytic fragments of alpha-actinin (27 and 53 kD) will be microinjected into myotubes in an attempt to interfere with the formation of premyofibrils and the transformation of premyofibrils into mature myofibrils. The second specific aim is to use a GFP targeting assay to determine which subdomains of the N-terminus of titin can localize to the Z-bands of skeletal muscle cells. We will probe the role of the different domains of this region of titin in targeting the Z-band and costameric proteins to the Z-bands. Protein-protein interaction assays will be used to identify the Z-band and costameric proteins, that interact with the Z-band regions of titin. The advanced optical methods coupled with molecular biological techniques will allow hypotheses about the formation of myofibrils to be tested directly in the single living myotube. These approaches should yield new insights into basic and pathologic processes in myofibril assembly in skeletal muscle cells.